The present invention relates to reagents for quantification of apolipoprotein B concentration in serum by enzyme immunoassay using sandwich method.
Hyperlipidemia plays a significant role in the causation of atherosclerosis. More specifically, an increase in the low-density lipoprotein (LDL) fractions or a decrease in the proportion of high-density lipoproteins (HDL) is considered to be an important indicator leading to atherosclerosis. While HDL-cholesterol measurement is generally used for clinical examination purposes, measurement of LDL-cholesterol or apolipoprotein B concentration is not in general use for such examination, because LDL measurement requires a prolonged ultra-centrifugation process.
In view of the fact that apolipoprotein B, as the major apoprotein component of LDL, represents as much as about 98% of the total apoprotein contents of LDL, the present inventors contemplated measuring apolipoprotein B concentration accurately and on a mass measurement basis to provide a marker as to the atherogenesity of LDL concentration.
In addition to that included in LDL, apolipoprotein B is partially contained in very-low-density lipoproteins (VLDL), but the apolipoprotein B content of VLDL is about 1/20 of that of LDL. Therefore, apolipoprotein B largely reflects LDL. In recent years, it has been recognized that VLDL and more particularly .beta.-VLDL are also contributory to atherosclerosis. As such, measurement of apolipoprotein B contained in both LDL and VLDL is therefore clinitically useful.
A highly sensitive measurement technique is also essential for detection of diseases involving decrease in LDL or VLDL concentration in plasma, such as lipoprotein B deficiency, liver diseases and intestinal deseases with absorption disorders.
At some facilities, techniques such as radial immunodiffusion (SRID) and rocket immunoelectrophoresis are employed in measuring apolipoprotein B concentration in serum. However, these methods have their disadvantages. The former method is rather low in sensitivity (30-200 mg/dl), and it requires visual evaluation which involves considerable variation according to the person who makes the determination. Therefore, it is unsuitable for multispecimen evaluation. The later method requires troublesome electrophoresis and it is rather complicated.
Another method known as radioimmunoassay (RIA) technique may be considered, but this method has not yet been developed well to practical application. Moreover, the fact that the method uses a radioactive isotope (.sup.125 I) involves various problems, such as radiation exposure and accumulation possibilities, a certain qualification required in practicing the method, expensive facility and equipment required, short half life of the isotope which means short service life thereof, possible environmental pollution, and limitations on waste disposal. Therefore, it is impractical to use the RIA technique for a regular clinical examination purposes.
As a measurement technique which involves no use of such radioactive material there is available a method known as enzyme immunoassay. However, no method of the type has yet been reported which can meet such practical requirements as high accuracy, good sensitivity, ease of control, and simplicity.
We have therefore developed an enzyme immunoassay which dissolves the above problems.
The present invention has as its object the provision of practically advantageous reagents for evaluation of apolipoprotein B concentration in human serum by enzyme immunoassay.